Defining the conformations of solution-phase Tau monomers and small aggregates using single-molecule spectroscopy Intrinsically disordered proteins, biomolecules possessing extremely fluid conformations that cannot be accurately described by a single static structure, play many roles in human disease. Tau, a largely structure-less microtubule-binding protein, lies at the heart of a variety of neuro-degenerative Tauopathies including Alzheimer?s disease. Critical outstanding questions include how these proteins lose their required functions, and form potentially toxic aggregates. However, even as recent cryo-EM data has revealed the molecular details of insoluble Tau aggregates, the molecular details of Tau monomers and small soluble aggregates remain unexplored. Because Tau can adopt many configurations and potentially form different structures, a well-suited probe method must be able to accurately quantify this diversity. Single-molecule methods, a class of techniques that entail the optical measurement of the properties of individual molecules, are uniquely suited to enable a quantitative description of Tau structure and behavior. The power of single-molecule methods stems from their ability to enable direct characterization of an inherently heterogeneous population of target molecules. However, existing single-molecule measurements require an untenable experimental compromise: molecules of interest must be probed only for several milliseconds, too short of a time period to allow observation of important dynamics and allow high-resolution characterization, or be immobilized in a manner that entails significant perturbations to inherent structure and behavior. Intrinsically disordered proteins like Tau are particularly susceptible to the highly perturbative influences of immobilization. A new single-molecule approach is required. We will use a microfluidic electrokinetic trap capable of cancelling Brownian motion in order to make statistically robust measurements on individual solution-phase molecules and small aggregates without the danger of perturbation from immobilization. Individual monomers and small aggregates will be targets of an investigation that relies on Forster Resonance Energy Transfer and Fluorescence Polarization Anisotropy to obtain molecular details on the structure of soluble Tau monomers and small aggregates. These details will be used to elucidate how the structure of Tau changes going from monomers to small oligomers to large aggregates, critical questions for understanding Tau?s mechanism and role in Alzheimer?s disease.